Enzyme stabilization

ABSTRACT

The glucose isomerase activity contained within bacterial cells can be stabilized by treating the bacterial cells with glutaraldehyde. The resulting stabilized enzyme can be used for conversion of glucose to fructose and then reused for further conversions with minimum reduction in enzyme activity after each use.

United States Patent [191 Zienty [22] Filed:

[73] Assignee: Miles Laboratories, Inc., Elkhart,

Ind.

May 13, 1971 211 Appl. No.: 143,194

[52] US. Cl. 195/68, 195/56 [5!] Int. Cl Cl2d 13/10 [58] Field of Search195/65, 66, 63, 68,

[56] References Cited UNITED STATES PATENTS 3,625,828 l2/l97l Brownewell195/66 R 3,694,314 9/1972 Lloyd et al. 195/31 F OTHER PUBLICATIONS Ellaret al., Chemical Abstracts, Vol. 74, 7I784X,

Ellar et al., Biochimica et Biophysica Acta, Vol. 225, pgs. 140-150,(1971).

Primary ExaminerLionel M. Shapiro Attorney-Joseph C. Schwalbach, LouisE. Davidson, Harry Stephenson and George R. Caruso [5 7] ABSTRACT Theglucose isomerase activity contained within bacterial cells can bestabilized by treating the bacterial cells with glutaraldehyde. Theresulting stabilized enzyme can be used for conversion of glucose tofructose and then reused for further conversions with minimum reductionin enzyme activity after each use.

3 Claims, N0 Drawings ENZYME STABILIZATION BACKGROUND AND PRIOR ART Itis known that a glucose isomerase enzyme can be employed to catalyze theconversion of glucose (dextrosc) to fructose (levulose) which has highersweetening power than the starting material. Glucose isomerase is alsoknown to be produced by fermentation of organisms, such as Pseudomonashydrophila, Streptomyces flavovireus, Streptomyces echinatur,Streptomyces achromogenus, Streptomyces albus, Streptomyces olivaceusand the like, in appropriate nutrient media. The glucose isomerase isformed inside the bacterial cells which grow during its production. Thecells can be filtered off from the fermentation beer and used directlyas a source of glucose isomerase. Alternatively, the cells can berecovered by filtration and then ruptured by well-known means. Theresulting ruptured cells and released ,contents can be used as a sourceof glucose isomerase. Still further, the cells can be ruptured and thedebris removed by centrifugation. The supernatant liquid can be useddirectly as a source of glucose isomerase or the enzyme can be recoveredas a powder from this liquid by well-known techniques. I

The glucose isomerase produced by all these prior art techniques did notbecome commercially useful because of the high cost of use of theenzyme. The enzyme production costs were high because of the expensivenutrient media necessary and the relatively low yields of enzyme ascompared to production techniques for other enzymes. It is thereforenecessary that the glucose isomerase be capable of being recovered afteruse and then reused for further conversions of glucose to fructose.

The glucose isomerase is most suitable for recovery and reuse if it isstill contained within the original bacterial cells. The bacterial cellscan be easily separated from the sugar-containing reaction conversionmedium. However, the prior art techniques still prohibited significantreuse of the bacterial cells since the cells lost about 50 percent oftheir glucose isomerase activity during each use.

SUMMARY OF THE INVENTION In accordance 'with the present invention, aprocess is provided for stabilization of glucose isomerase in bacterialcells which comprises treating bacterial cells containing glucoseisomerase activity with glutaraldehyde.

DESCRIPTION OF THE INVENTION The bacterial cells containing glucoseisomerase activity useful in the process of the present invention can beproduced by well-known procedures. The preferred enzyme-containing cellsare produced by growing under submerged aerobic conditions a culture ofStreptomyces alivaceus NRRL 3583 or mutants thereof in a mediumcontaining appropriate nutrients. The resulting bacterial cells areseparated from the fermentation beer by filtration or centrifugation.

The recovered bacterial cells are then suspended in an aqueous mediumand are mixed with glutaraldehyde. The glutaraldehyde is employed in anamount from about 0.1 to about 50 weight percent based on the dry weightof the cells. Preferably, the glutaraldehyde is employed in an amountfrom about to about 50 weight percent based on the dry weight of thecells. The

bacterial cells initially have a pH of about 8.5. As the bacterial cellsare treated with the glutaraldehyde, the pH drops eventually to about6.5. If the initial pH is above about 8.5 or if the pH during treatmentdrops to below about 6.5, suitable buffering materials should be addedto maintain the pH of the bacterial cells within the range from about6.5 to about 8.5.

The bacterial cells should be treated with the glutaraldehyde for fromabout one-half to about 2 hours. The preferred treatment time is fromabout 1 to about 1% hours.

The treatment temperature is not narrowly critical and can convenientlybe from about 15C. to about 60C.

The glucose isomerase activity of the starting material bacterial cellsand the glutaraldehyde-treated material was assayed by the followingprocedure.

GLUCOSE ISOMERASE ASSAY A 62.5 percent (weight/volume basis) aqueoussolution of glucose was prepared by slowly adding with stirring 625grams of anhydrous glucose to 300 ml. of hot distilled water. Thesolution was cooled'to room temperature and ml. of 1 molar concentrationaqueous solution of tris (hydroxymethyl) aminoethane at pH 8.5, 125 ml.of 1 molar concentration aqueous magnesium sulfate solution and 50 ml.of 0.025 molar concentration aqueous cobaltous chloride solution wereadded. The resulting mixture was then diluted to 1 liter with distilledwater.

A 10 ml. portion of the above glucose solution was added to a 25 ml.volumetric flask. Sufficient enzyme to be assayed was added to produce areduction of specific rotation, as hereinafter defined, of about 2.9 toabout 7.5". The flask contents were then diluted to 25 ml. withdistilled water, and the resulting mixture was incubated at 60C. for 2hours. The reaction was then stopped by adding 1 ml. of a 0.5 molarconcentration aqueous solution of perchloric acid. The mixture was thencentrifuged at 15,000 R.P.M. for 20 minutes, and

the optical rotation (in degrees) of the supernatant liquid was measuredby well-known techniques. A blank was run by the same procedure butwithoutthe presence of the enzyme. The optical rotation. of the blankwas also measured. The specific rotation, {011 of the sample or blankwas 204 or two times the observed optical rotation. The percentconversion of glucose to fructose was calculated by the followingformula:

Percent glucose conversion [ah [a],,,,,,,,,,.

[[al l X The enzyme activity in glucose isomerase units (GIU) of theenzyme sample being assayed was calculated by the following formula:

GIU/gram or GlU/liter micrograms fructose formed X 0.0463 /mg. or ml.enzyme used wherein the micrograms fructose formed were calculated bymultiplying the above-calculated Percent Glucose Conversion value by6.25 X 10. A glucose isomerase unit is the amount of enzyme which willconvert one micromole of glucose to fructose per minute under theconditions of the assay.

The invention will be described in further detail in the followingexamples.

EXAMPLE 1 A culture of Streptomyces olivaceus NRRL 3583 was transferredto an agitated aerated fermentor containing liters of an aqueous mixturecontaining 0.7 percent xylose, 0.3 percent refined corn starch, 1.0percent peptone, 0.50 percent meat extract, 0.25 percent yeast extract,0.50 percent sodium chloride and 0.05 percent magnesium sulfate andhaving a pH of 7.0. All the above percent values were on a weight/volumebasis. The agitator was rotated at 400 R.P.M. and air was passed throughthe medium at a rate of 3 volumes of air per volume of medium perminute. The fermentation was continued for 24 hours at 32C. Thefermentation beer was then centrifuged at 40,000 R.P.M. to separate thebacterial cells. A portion of the bacterial cells was then frozen andlyophilized.

Three grams of the freeze-dried whole cells were suspended in 50 ml. ofaqueous tris (hydroxymethyl) aminomethane at pH 8.5, and the stirredsuspension was treated with 0.1 gm. of a 25 weight percent aqueousglutaraldehyde solution (0.83 weight percent glutaraldehyde based on dryweight of the cells) with stirring at room temperature (about C.) for1.5 hours. The treated cells having an activity of 145 GIU/gram werecollected by filtration and added to a 62.5 weight percent aqueoussolution of glucose containing 0.001 weight percent cobalt chloride and0.01 weight percent magnesium chloride. The resulting mixture wasreacted at 70C. for 2 hours to convert about 4.5 percent of the glucoseto fructose. The cells were separated from the reaction mixture bycentrifugation and the fructosedextrose mixture was decanted. The cellswere assayed to have the same glucose isomerase activity as before theiruse. The cells were then used again to treat a fresh glucose solutionunder the same conditions as described above and were recovered andassayed. This procedure was repeated for a total of twelve uses of thecells. These cells still contained 56 percent of the initial glucoseisomerase activity. Cells which had not been treated with glutaraldehydelost substantially all their glucose isomerase activity upon being usedtwice.

EXAMPLE 2 A fermentation beer containing Streptomyces olivaceus NRRL3583 bacterial cells was prepared in the manner described in Example 1.A portion of this beer was centrifuged and a 3 gram portion of wet wholebacterial cells was isolated. These cells were then suspended in 50 ml.of water and treated with 0.1 gm. of a weight percent aqueousglutaraldehyde solution in the pH range of 6.5 to 8.5 at roomtemperature for 1 hour. The treated cells having an activity of 265GlUlgram were collected by filtration and were used to convert glucoseto fructose in the manner described in Example 1. The cells wererecovered and reused with fresh glucose for a total of twelve times. Thecells still retained 60.7 percent of the initial glucose isomeraseactivity.

EXAMPLE 3 A fermentation beer containing Srreptomyces olivaceus NRRL3583 bacterial cells was prepared in the manner described in Example 1.An aliquot portion of the whole beer containing 3 gm. of whole cells wasmixed with 0.2 gm. of a 25 weight percent aqueous glutaraldehydesolution (1.67 weight percent glutaraldehyde based on dry weight of thecells) with stirring at room temperature and in the pH range of 6.5 to8.5 for 1 hour. The treated cells having an activity of 271 G1- U/gramwere collected by filtration and were used to convert a glucose tofructose in the manner described in Example 1. The cells were recoveredand reused with fresh glucose for a total of twelve times. The cellsstill retained 61.6 percent of the initial glucose isomerase activity.

EXAMPLE 4 A fermentation beer containing Streptomyces olivaceus NRRL3583 bacterial cells was prepared in the manner described in Example 1.An aliquot portion of the whole beer containing 4 gm. of whole cells wasmixed with 0.4 gm. ofa 25 weight percent aqueous glutaraldehyde solution(2.5 weight percent glutaraldehyde based on dry weight of the cells)with stirring at room temperature for 1 hour. No pH control wasemployed. At the start of the reaction the cell mixture had a pH of 8.4.At the end of the reaction period, the pH was 6.8. The treated cellshaving an activity of 387 Gl- U/gram were collected by filtration andwere used to convert glucose to fructose in the manner described inExample 1. The cells were recovered and reused with fresh glucose for atotal of five times. The cells still retained 100 percent of the initialglucose isomerase activity.

EXAMPLE 5 A fermentation beer containing Streptomyces olivaceus NRRL3583 bacterial cells was prepared in the manner described in Example 1employing a 100 gal. fermentor. This beer had a total activity of1,350,000 GlU and contained 7 lbs. of bacterial cells on a dry weightbasis (425 GlU/gm.). The pH of the beer was adjusted to 8.2 by additionof sodium hydroxide. A 7 lb. portion of 50 weight percent aqueousglutaraldehyde solution diluted to a concentration of 0.5-0.6 percent(weight/volume basis) was added to the beer with agitation over a 30-40min. period (50 weight percent glutaraldehyde based on dry weight ofcells). The resulting mixture was then reacted at room temperature for1.5 hr. with mild agitation. A 0.5 percent (weightlvolume basis) portionof diatomaceous earth filter aid was then added and the beer wasfiltered on a rotary vacuum filter. The filter cake was washed withwater to remove spent beer residues. The collected bacterial cells had atotal activity of 1,215,000 GlU or percent of the initial activity.

A glucose solution was prepared by mixing 225 lb. of glucose in 55 gal.of water (49.2 percent glucose on a weight/volume basis). To thissolution were added cobalt chloride in an amount of 0.001 percent(weightlvolume basis) and magnesium chloride in an amount of 0.01percent (weight/volume basis). A portion of the above-preparedglutaraldehyde-treated bacterial cells having a total activity of407,250 GlU was also added to the glucose solution. The resultingmixture was stirred at 60C. for 24 hr. during which time the pH wasmaintained at 7.7-7.9 by addition of sodium hydroxide. The bacterialcells were filtered off from the reaction mixture which was then assayedto contain 40.2 percent fructose. The above-collected bacterial cellswere then reused on a fresh glucose solution in the manner describedabove. This procedure was then repeated until the bacterial cells hadbeen used a total of nine 'from about 6.5 to about 8.5 with from about0.1 to

about 50 weight percent glutaraldehyde based on the dry weight of thecells.

2. A process according to claim 1 wherein the glutaraldehyde is employedin an amount from about 10 to about 50 weight percent based on the dryweight of the cells.

3. A process according to claim 1 wherein the bacterial cells areproduced from Streptomyces olivaceus.

2. A process according to claim 1 wherein the glutaraldehyde is employedin an amount from about 10 to about 50 weight percent based on the dryweight of the cells.
 3. A process according to claim 1 wherein thebacterial cells are produced from Streptomyces olivaceus.